The present invention relates to compounds, compositions, the preparation of such compounds and methods for the treatment of hepatitis C virus (HCV) infection. In particular, the present invention provides novel peptide analogues, pharmaceutical compositions containing such analogues and methods for using these analogues in the treatment of HCV infection.
Hepatitis C virus (HCV) is the major etiological agent of post-transfusion and community-acquired non-A non-B hepatitis worldwide. It is estimated that over 170 million people worldwide are infected by the virus. A high percentage of carriers become chronically infected and many progress to chronic liver disease, so-called chronic hepatitis C. This group is in turn at high risk for serious liver disease such as liver cirrhosis, hepatocellular carcinoma and terminal liver disease leading to death.
The mechanism by which HCV establishes viral persistence and causes a high rate of chronic liver disease has not been thoroughly elucidated. It is not known how HCV interacts with and evades the host immune system. In addition, the roles of cellular and humoral immune responses in protection against HCV infection and disease have yet to be established. Immunoglobulins have been reported for prophylaxis of transfusion-associated viral hepatitis, however, the Center for Disease Control does not presently recommend immunoglobulins treatment for this purpose. The lack of an effective protective immune response is hampering the development of a vaccine or adequate post-exposure prophylaxis measures, so in the near-term, hopes are firmly pinned on antiviral interventions. Various clinical studies have been conducted with the goal of identifying pharmaceutical agents capable of effectively treating HCV infection in patients afflicted with chronic hepatitis C. These studies have involved the use of interferon-alpha, alone and in combination with other antiviral agents. Such studies have shown that a substantial number of the participants do not respond to these therapies, and of those that do respond favorably, a large proportion were found to relapse after termination of treatment.
Until a few years ago, interferon (IFN) was the only available therapy of proven benefit approved in the clinic for patients with chronic hepatitis C. However the sustained response rate is low, and interferon treatment also induces severe side-effects (i.e. retinopathy, thyroiditis, acute pancreatitis, depression) that diminish the quality of life of treated patients. Interferon in combination with ribavirin was originally approved for patients non-responsive to IFN alone. It has now been approved for naive patients and presently constitutes the gold standard in HCV therapy. However, the side effects caused by IFN are not alleviated with this combination therapy.
Therefore, a need exists for the development of effective antiviral agents for treatment of HCV infection that overcomes the limitations of existing pharmaceutical therapies.
HCV is an enveloped positive strand RNA virus in the Flaviviridae family. The single strand HCV RNA genome is approximately 9500 nucleotides in length and has a single open reading frame (ORF) encoding a single large polyprotein of about 3000 amino acids. In infected cells, this polyprotein is cleaved at multiple sites by cellular and viral proteases to produce the structural and non-structural (NS) proteins. In the case of HCV, the generation of mature nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) is effected by two viral proteases. The first one, as yet poorly characterized, cleaves at the NS2-NS3 junction; the second one is a serine protease contained within the N-terminal region of NS3 (henceforth referred to as NS3 protease) and mediates all the subsequent cleavages downstream of NS3, both in cis, at the NS3-NS4A cleavage site, and in trans, for the remaining NS4A-NS4B, NS4B-NS5A, NS5A-NS5B sites. The NS4A protein appears to serve multiple functions, acting as a cofactor for the NS3 protease and possibly assisting in the membrane localization of NS3 and other viral replicase components. The complex formation of the NS3 protein with NS4A seems necessary to the processing events, enhancing the proteolytic efficiency at all of the sites. The NS3 protein also exhibits nucleoside triphosphatase and RNA helicase activities. NS5B is a RNA-dependent RNA polymerase that is involved in the replication of HCV.
Patent application WO 97/06804 describes the (xe2x88x92) enantiomer of the nucleoside analogue cytosine-1,3-oxathiolane (also known as 3TC) as active against HCV. This compound, although reported as safe in previous clinical trials against HIV and HBV, has yet to be clinically proven active against HCV and its mechanism of action against the virus has yet to be reported.
A general strategy for the development of antiviral agents is to inactivate virally encoded enzymes that are essential for the replication of the virus.
In this vein, intense efforts to discover compounds which inhibit the NS3 protease or RNA helicase of HCV have led to the following disclosures:
U.S. Pat. No. 5,633,388 describes heterocyclic-substituted carboxamides and analogues as being active against HCV. These compounds are directed against the helicase activity of the NS3 protein of the virus but clinical tests have not yet been reported. A phenanthrenequinone has been reported by Chu et al., (Tet. Lett., (1996), 7229-7232) to have activity against the HCV NS3 protease in vitro. No further development on this compound has been reported.
A paper presented at the Ninth International Conference on Antiviral Research, Urabandai, Fukyshima, Japan (1996) (Antiviral Research, (1996), 30, 1, A23 (abstract 19)) reports thiazolidine derivatives to be inhibitory to the HCV protease.
Several studies have reported compounds inhibitory to other serine proteases, such as human leukocyte elastase. One family of these compounds is reported in WO 95/33764 (Hoechst Marion Roussel, 1995). The peptides disclosed in that application are morpholinylcarbonyl-benzoyl-peptide analogues that are structurally different from the peptides of the present invention.
WO 98/17679 from Vertex Pharmaceuticals Inc. discloses inhibitors of serine protease, particularly, Hepatitis C virus NS3 protease Hoffman LaRoche (WO 98/22496; U.S. Pat. Nos. 5,866,684 and 6,018,020) has also reported hexapeptides that are proteinase inhibitors useful as antiviral agents for the treatment of HCV infection.
Steinkxc3xchler et al. and Ingallinella et al. have published on NS4A-4B product inhibition (Biochemistry (1998), 37, 8899-8905 and 8906-8914).
WO 97/43310 by Schering Corporation discloses 20 and 21 amino acid peptide sequences active against the HCV NS3 protease.
WO 98/46597 by Emory University discloses peptides and peptidomimetics active in vitro against serine proteases.
WO 98/46630 by Peptide Therapeutics Limited discloses depsipeptide substrate inhibiting the HCV NS3 protease.
Finally, U.S. Pat. No. 5,869,253 discloses enzymatic RNA molecules that inhibit the HCV NS3 protease.
None of the prior patent applications described above disclose suggest cyclic peptides active and selective against the Hepatitis C virus NS3 protease.
WO 99/07733, WO 99/07734, WO 00/09543 and WO00/09558 disclose hexa to tetra-peptides and tripeptide analogs that inhibit the NS3 protease. However, these disclosures do not suggest or lead to the design of macrocyclic analogs of the present invention.
WO 99/38888 published Aug. 5, 1999 by the Institute de Richerche di Biologia Moleculare (IRBM) discloses small peptides inhibitors of the HCV NS3 protease. Nothing in this disclosure suggest or indicates the cyclic nature of the peptides of the present invention. In addition, this PCT application was published after the priority date of the present application.
WO 99/64442 by IRBM, also published after the priority date of this application, discloses oligopeptides with ketoacids at P1.
WO 99/50230 from Vertex Pharmaceuticals (published on Oct. 7, 1999) was also published after the priority date of the present application. Even then, the publication does not remotely suggest any cyclic peptides of the present invention.
One advantage of the present invention is that it provides macrocyclic peptides that are inhibitory to the NS3 protease of the hepatitis C virus.
A further advantage of one aspect of the present invention resides in the fact that these peptides specifically inhibit the NS3 protease and do not show significant inhibitory activity against other serine proteases such as human leukocyte elastase (HLE), porcine pancreatic elastase (PPE), or bovine pancreatic chymotrypsin, or cysteine proteases such as human liver cathepsin B (Cat B).
A further advantage of the present invention is that it provides small peptides of low molecular weight that are capable of penetrating cell membranes and inhibit the NS3 protease activity in cell culture.
Still, a further advantage of the compounds of the present invention resides in the fact that they are active in both major genotypes found in clinical isolates (1a and 1b), strongly suggesting that these compound will be active against all presently known genotypes of HCV.
Included in the scope of the invention are compounds of formula (I): 
wherein
W is CH or N,
R21 is H, halo, C1-6 alkyl, C3-6 cycloalkyl, C1-6 haloalkyl, C1-6 alkoxy, C3-6 cycloalkoxy, hydroxy, or N(R23)2, wherein each R23 is independently H, C1-6 alkyl or C3-6 cycloalkyl; and
R22 is H, halo, C1-6 alkyl, C3-6 cycloalkyl, C1-6 haloalkyl, C1-6 thioalkyl, C1-6 alkoxy, C3-6 cycloalkoxy, C2-7 alkoxyalkyl, C3-6 cycloalkyl, C6 or 10 aryl or Het, wherein Het is a five-, six-, or seven-membered, saturated or unsaturated heterocycle, containing from one to four heteroatoms selected from nitrogen, oxygen and sulfur;
said cycloalkyl, aryl or Het being substituted with R24,
wherein R24 is H, halo, C1-6 alkyl, C3-6 cycloalkyl, C1-6 alkoxy, C3-6 cycloalkoxy, NO2, N(R25)2; NHxe2x80x94C(O)xe2x80x94R25, or NHxe2x80x94C(O)xe2x80x94NHxe2x80x94R25,
wherein each R25 is independently: H, C1-6 alkyl or C3-6 cycloalkyl;
or R24 is NHxe2x80x94C(O)xe2x80x94OR26 wherein R26 is C1-6 alkyl or C3-6 cycloalkyl;
R3 is hydroxy, NH2, or a group of formula xe2x80x94NHxe2x80x94R31, wherein R31 is C6 or 10 aryl, heteroaryl, xe2x80x94C(O)xe2x80x94R32, xe2x80x94C(O)xe2x80x94OR32, or xe2x80x94C(O)xe2x80x94NHR32,
wherein R32 is: C1-6 alkyl or C3-6 cycloalkyl;
D is a 5 to 10-atom saturated or unsaturated alkylene chain optionally containing one to three heteroatoms independently selected from: O, S, or Nxe2x80x94R41, wherein
R41 is H, C1-6 alkyl, C3-6 cycloalkyl, or xe2x80x94C(O)xe2x80x94R42, wherein R42 is C1-6 alkyl, C3-6 cycloalkyl or C6 or 10 aryl; and wherein the atoms of the D chain that form part of the macrocyclic ring in structural formula (I) are numbered from left to right in structural formula (I) starting with position number 8.
R4 is H or from one to three substituents at any carbon atom of said chain D, said substituent independently selected from the group consisting of: C1-6 alkyl, C1-6 haloalkyl, C1-6 alkoxy, hydroxy, halo, amino, oxo, thio, or C1-6 thioalkyl and
A is an amide of formula xe2x80x94C(O)xe2x80x94NHxe2x80x94R5, wherein R5 is selected from the group consisting of: C1-8 alkyl, C3-6 cycloalkyl, C6 or 10 aryl or C7-16 aralkyl;
or A is a carboxylic acid or a pharmaceutically acceptable salt or ester thereof.
Included within the scope of this invention is a pharmaceutical composition comprising an anti-hepatitis C virally effective amount of a compound of formula I, or a therapeutically acceptable salt or ester thereof, in admixture with a pharmaceutically acceptable carrier medium or auxiliary agent.
An important aspect of the invention involves a method of treating a hepatitis C viral infection in a mammal by administering to the mammal an anti-hepatitis C virally effective amount of the compound of formula I, or a therapeutically acceptable salt or ester thereof or a composition as described above.
Another important aspect involves a method of inhibiting the replication of hepatitis C virus by exposing the virus to a hepatitis C NS3 protease-inhibiting amount of the compound of formula I, or a therapeutically acceptable salt or ester thereof or a composition as described above.
Still another aspect involves a method of treating a hepatitis C viral infection in a mammal by administering thereto an anti-hepatitis C virally effective amount of a combination of the compound of formula I, or a therapeutically acceptable salt or ester thereof. According to one embodiment, the pharmaceutical compositions of this invention comprise an additional immunomodulatory agent. Examples of additional immunomodulatory agents include but are not limited to, xcex1-, xcex2-, and xcex4-interferons.
According to an alternate embodiment, the pharmaceutical compositions of this invention may additionally comprise an antiviral agent. Examples of antiviral agents include, ribavirin and amantadine.
According to another alternate embodiment, the pharmaceutical compositions of this invention may additionally comprise other inhibitors of HCV protease.
According to yet another alternate embodiment, the pharmaceutical compositions of this invention may additionally comprise an inhibitor of other targets in the HCV life cycle, such as helicase, polymerase, metalloprotease or IRES.
Definitions
As used herein, the following definitions apply unless otherwise noted:
The designation herein of a position within the D chain by position number(s), e.g. xe2x80x9cposition 10 of said D chainxe2x80x9d or xe2x80x9cD chain is substituted at position 8xe2x80x9d or xe2x80x9cdouble bond at position 13, 14 of said D chainxe2x80x9d or xe2x80x9cD chain contains one double bond at position 11,12xe2x80x9d, or similar language, means the position or positions within the D chain when the atoms of the D chain are numbered as set forth previously, i.e., the atoms of the D chain that form part of the macrocyclic ring in structural formula (I) are numbered from left to right in structural formula (I) starting with position number 8.
With reference to the instances where (R) or (S) is used to designate the absolute configuration of a substituent, e.g. R4 of the compound of formula I, the designation is done in the context of the whole compound and not in the context of the substituent alone.
The designation xe2x80x9cP1, P2, and P3xe2x80x9d as used herein refer to the position of the amino acid residues starting from the C-terminus end of the peptide analogs and extending towards the N-terminus (i.e. P1 refers to position 1 from the C-terminus, P2: second position from the C-terminus, etc.) (see Berger A. and Schechter I., Transactions of the Royal Society London series B257, 249-264 (1970)).
As used herein the term xe2x80x9c1-aminocyclopropyl-carboxylic acidxe2x80x9d (ACCA) refers to a compound of formula: 
As used herein the term xe2x80x9cvinyl-ACCAxe2x80x9d refers to a compound of formula: 
As used herein the term xe2x80x9chomo-allyl-ACCAxe2x80x9d refers to a compound of formula: 
The term xe2x80x9chaloxe2x80x9d as used herein means a halogen substituent selected from bromo, chloro, fluoro or iodo.
The term xe2x80x9cC1-6 haloalkylxe2x80x9d as used herein means as used herein, either alone or in combination with another substituent, means acyclic, straight or branched chain alkyl substituents containing from 1 to six carbon atoms having one or more hydrogen substituted for a halogen selected from bromo, chloro, fluoro or iodo.
The term xe2x80x9cC1-6 thioalkylxe2x80x9d as used herein means as used herein, either alone or in combination with another substituent, means acyclic, straight or branched chain alkyl substituents containing a thiol group such a thiopropyl.
The term xe2x80x9cC1-6 alkylxe2x80x9d or xe2x80x9c(lower)alkylxe2x80x9d as used herein, either alone or in combination with another substituent, means acyclic, straight or branched chain alkyl substituents containing from 1 to six carbon atoms and includes, for example, methyl, ethyl, propyl, butyl, hexyl, 1-methylethyl, 1-methylpropyl, 2-methylpropyl, 1,1-dimethylethyl.
The term xe2x80x9cC3-6 cycloalkylxe2x80x9d as used herein, either alone or in combination with another substituent, means a cycloalkyl substituent containing from three to six carbon atoms and includes cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.
The term xe2x80x9cunsaturated cycloalkylxe2x80x9d includes, for example, the substituent cyclohexenyl: 
The term xe2x80x9csaturated or unsaturated alkylenexe2x80x9d as used herein means a divalent alkyl substituent derived by the removal of one hydrogen atom from each end of a saturated or unsaturated straight or branched chain aliphatic hydrocarbon and includes, for example, xe2x80x94CH2CH2C(CH3)2CH2CH2xe2x80x94, xe2x80x94CH2CH2CHxe2x95x90CHCH2CH2xe2x80x94 or xe2x80x94CH2Cxe2x89xa1xe2x80x94CCH2CH2xe2x80x94. This alkyl chain may optionally contain a heteroatom such as oxygen (for example: CH3xe2x80x94CH2xe2x80x94Oxe2x80x94CH2xe2x80x94).
The term xe2x80x9cC1-6 alkoxyxe2x80x9d as used herein, either alone or in combination with another substituent, means the substituent xe2x80x94Oxe2x80x94C1-6 alkyl wherein alkyl is as defined above containing up to six carbon atoms. Alkoxy includes methoxy, ethoxy, propoxy, 1-methylethoxy, butoxy and 1,1-dimethylethoxy. The latter substituent is known commonly as tert-butoxy.
The term xe2x80x9cC3-6 cycloalkoxyxe2x80x9d as used herein, either alone or in combination with another substituent, means the substituent xe2x80x94Oxe2x80x94C3-6 cycloalkyl containing from three to 6 carbon atoms.
The term xe2x80x9cC1-6 alkoxyalkylxe2x80x9d as used herein, means the substituent C1-6 alkyl-Oxe2x80x94C1-6 alkyl wherein alkyl is as defined above containing up to six carbon atoms. For example, methoxymethyl means xe2x80x94CH2xe2x80x94Oxe2x80x94CH3.
The term xe2x80x9cC2-7 acylxe2x80x9d as used herein, either alone or in combination with another substituent, means an C1-6 alkyl group linked through a carbonyl group such as xe2x80x94C(O)xe2x80x94C1-6 alkyl.
The term xe2x80x9cC6 or C10 arylxe2x80x9d as used herein, either alone or in combination with another substituent, means either an aromatic monocyclic system containing 6 carbon atoms or an aromatic bicyclic system containing 10 carbon atoms. For example, aryl includes a phenyl or a naphthylxe2x80x94ring system.
The term xe2x80x9cC7-16 aralkylxe2x80x9d as used herein, either alone or in combination with another substituent, means an aryl as defined above linked through an alkyl group, wherein alkyl is as defined above containing from 1 to 6 carbon atoms. Aralkyl includes for example benzyl, and butylphenyl.
The term xe2x80x9cHetxe2x80x9d as used herein, either alone or in combination with another substituent, means a monovalent substituent derived by removal of a hydrogen from a five-, six-, or seven-membered saturated or unsaturated (including aromatic) heterocycle containing from one to four heteroatoms selected from nitrogen, oxygen and sulfur. Examples of suitable heterocycles include: tetrahydrofuran, thiophene, diazepine, isoxazole, piperidine, dioxane, morpholine, pyrimidine or 
The term xe2x80x9cHetxe2x80x9d also includes a heterocycle as defined above fused to one or more other cycle be it a heterocycle or any other cycle. One such examples includes thiazolo[4,5-b]-pyridine.
Although generally covered under the term xe2x80x9cHetxe2x80x9d, the term xe2x80x9cheteroarylxe2x80x9d as used herein precisely defines an unsaturated heterocycle which is an aromatic system. Suitable example of heteroaromatic system include: quinoline, indole, pyridine, 
The term xe2x80x9cpharmaceutically acceptable esterxe2x80x9d as used herein, either alone or in combination with another substituent, means esters of the compound of formula I in which any of the carboxyl functions of the molecule, but preferably the carboxy terminus, is replaced by an alkoxycarbonyl function: 
in which the R moiety of the ester is selected from alkyl (e.g. methyl, ethyl, n-propyl, t-butyl, n-butyl); alkoxyalkyl (e.g. methoxymethyl); alkoxyacyl (e.g. acetoxymethyl); aralkyl (e.g. benzyl); aryloxyalkyl (e.g. phenoxymethyl); aryl (e.g. phenyl), optionally substituted with halogen, C1-4 alkyl or C1-4 alkoxy. Other suitable prodrug esters are found in Design of prodrugs, Bundgaard, H. Ed. Elsevier (1985) incorporated herewith by reference. Such pharmaceutically acceptable esters are usually hydrolyzed in vivo when injected in a mammal and transformed into the acid form of the compound of formula I.
With regard to the esters described above, unless otherwise specified, any alkyl moiety present advantageously contains 1 to 16 carbon atoms, particularly 1 to 6 carbon atoms. Any aryl moiety present in such esters advantageously comprises a phenyl group.
In particular the esters may be a C1-16 alkyl ester, an unsubstituted benzyl ester or a benzyl ester substituted with at least one halogen, C1-6 alkyl, C1-6 alkoxy, nitro or trifluoromethyl.
The term xe2x80x9cpharmaceutically acceptable saltxe2x80x9d as used herein includes those derived from pharmaceutically acceptable bases. Examples of suitable bases include choline, ethanolamine and ethylenediamine. Na+, K+, and Ca++ salts are also contemplated to be within the scope of the invention (also see Pharmaceutical salts, Birge, S. M. et al., J. Pharm. Sci., (1977), 66, 1-19, incorporated herein by reference).
Preferred Embodiments
R1 
Preferred embodiments of the present invention include compounds of formula I as described above, wherein the R1 moiety is selected from the 2 different diastereoisomers where the 1-carbon center has the R configuration as represented by structures (i) and (ii): 
More preferably, the linker D is linked to R1 in the configuration syn to A as represented by structure (ii).
R2 
Preferred embodiments of the present invention include compounds of formula I as described above, wherein the R2 moiety is 
wherein W is preferably N.
Preferably, R21 is H, C1-6 alkyl, C1-6 alkoxy, hydroxy, chloro, or N(R23)2 wherein R23 is preferably H or C1-6 alkyl. More preferably, R21 is H or C1-6 alkoxy. Most preferably, R21 is methoxy.
Preferably R22 is H, C1-6 thioalkyl, C1-6 alkoxy, phenyl or Het selected from the group consisting of: 
Preferably, R24 is H, C1-6 alkyl, NHxe2x80x94R25, NHxe2x80x94C(O)xe2x80x94R25; or NHxe2x80x94C(O)xe2x80x94NHxe2x80x94R25 or NHxe2x80x94C(O)xe2x80x94OR26.
More preferably R22 is C1-4 alkoxy, phenyl or Het selected from the group consisting of: 
More preferably, R24 is H, C1-6 alkyl, NHxe2x80x94R25, NHxe2x80x94C(O)xe2x80x94R25; or NHxe2x80x94C(O)xe2x80x94OR26.
Most preferably R22 is ethoxy, or Het selected from the group consisting of: 
Most preferably, R24a is NHxe2x80x94R25, NHxe2x80x94C(O)xe2x80x94R25, or NHxe2x80x94C(O)xe2x80x94OR26. Most preferably, R24b is H or C1-6 alkyl.
Preferably, each R25 is independently: H, C1-6 alkyl, or C3-6 cycloalkyl. More preferably, R25 is C1-6 alkyl or C3-6 cycloalkyl. More preferably, R25 is C1-6 alkyl.
Preferably, R26 is C1-6 alkyl.
R3 
Preferred embodiments of the present invention include compounds of formula I as described above, wherein the R3 moiety is preferably an amide of formula NHxe2x80x94C(O)xe2x80x94R32, a urea of formula NHxe2x80x94C(O)xe2x80x94NHxe2x80x94R32, or a carbamate of formula NHxe2x80x94C(O)xe2x80x94OR32. More preferably, R3 is a carbamate or a urea. Most preferably, R3 is a carbamate.
Preferably, R32 is C1-6 alkyl, or C3-6 cycloalkyl. More preferably, R32 is C1-6 alkyl, or C4-6 cycloalkyl. Most preferably, R32 is tert-butyl, cyclobutyl or cyclopentyl.
D
Preferred embodiments of the present invention include compounds of formula I, wherein linker D is a 6 to 8 atom saturated or unsaturated alkylene chain. More preferably, linker D is 7 atom chain.
Preferably, the D chain contains one or two heteroatoms selected from: O, S, NH, Nxe2x80x94C1-6 alkyl or Nxe2x80x94C2-7 acyl. More preferably, the D chain optionally contains one heteroatom selected from: NH, or Nxe2x80x94C2-7 acyl, most preferably N(Ac), and is positioned at atom 10 of the chain. Most preferably, the chain containing a nitrogen atom is saturated.
Alternatively, D contains one heteroatom selected from: O, or S. Preferably, when D is 7 atom in length, the O or S atom is at position 9 of the chain. Preferably, this chain is substituted with R4, wherein R4 is H or C1-6 alkyl. More preferably, R4 is H or methyl. Most preferably, R4 is H or 8-(S)xe2x80x94Me. Even most preferably, D is saturated. Alternatively, D contains one double bond at position 11,12. Preferably, this double bond is trans.
Alternatively, D is an all carbon saturated or unsaturated alkylene chain. In this case, D is preferably saturated and is 7 atom in length. More preferably, D is substituted with R4, wherein R4 is H, oxo, thio, hydroxy, thioalkyl, alkoxy or alkyl. More preferably, R4 is H or C1-6 alkyl. Most preferably, R4 is H or methyl. Most preferably, R4 is H or 10-(S)xe2x80x94Me.
Alternatively, D is an all carbon alkylene chain containing preferably one double bond and is 7 atom in length. More preferably, this double bond is at position 13,14 of the chain. Most preferably, this double bond is cis. Preferably, this D chain is substituted with R4, wherein R4 is H, oxo, hydroxy, alkoxy or alkyl. More preferably, R4 is H or C1-6 alkyl. Even more preferably, R4 is H or methyl. Most preferably, R4 is H or 10-(S)xe2x80x94Me.
A
Preferred embodiments of the present invention include compounds of formula I as described above, wherein A is a carboxylic acid.
Specific Embodiments
Preferred embodiments of the present invention include compounds of formula I as described above, wherein R2 is a quinoline substituent (i.e. W is N);
R3 is a group of formula xe2x80x94NHxe2x80x94C(O)xe2x80x94NHR32 or xe2x80x94NHxe2x80x94C(O)xe2x80x94OR32,
wherein R32 is: C1-4 alkyl or C4-6 cycloalkyl;
D is a 6 to 8 atom saturated or unsaturated alkylene chain linked to R1 in configuration syn to A, optionally containing one or two heteroatoms independently selected from: O, S or Nxe2x80x94R41, wherein R41 is C2-7 acyl;
R4 is H, or from one to three substituents independently selected from hydroxy or C1-6 alkyl; and
A is a carboxylic acid, or a pharmaceutically acceptable salt or ester thereof.
More preferably are compounds of formula I wherein R1 is as defined above; R21 is H or methoxy;
R22 is C1-6 alkoxy, or Het selected from the group consisting of: 
xe2x80x83wherein
R24a is H, C1-6 alkyl, NHxe2x80x94R25, NHxe2x80x94C(O)xe2x80x94R25, NHxe2x80x94C(O)xe2x80x94NHxe2x80x94R25,
wherein R25 is: H, C1-6 alkyl or C3-6 cycloalkyl;
or R24a is NHxe2x80x94C(O)xe2x80x94OR26, wherein R26 is C1-6 alkyl or C3-6 cycloalkyl;
and R24b is H or C1-6 alkyl;
R3 is a urea of the formula NHxe2x80x94C(O)xe2x80x94NHR32 or a carbamate of formula NHxe2x80x94C(O)xe2x80x94OR32, wherein R32 is C1-6 alkyl or C3-6 cycloalkyl;
D is a C7-atom saturated or unsaturated alkylene chain optionally containing one double bond at position 11,12 or 13,14;
said D chain optionally containing one heteroatom independently selected from: O, S, NH, N(Me), or N(Ac); and
R4 is H or C1-6 alkyl.
Most preferably, are compounds of formula I wherein R21 is methoxy, and R22 is ethoxy or: 
wherein
R24a is NHxe2x80x94(C1-4 alkyl), NHxe2x80x94C(O)xe2x80x94(C1-4 alkyl); or NHxe2x80x94C(O)xe2x80x94Oxe2x80x94(C1-4 alkyl),; and
D is saturated or contains one cis double bond at position 13,14.
Finally, included within the scope of this invention are all compounds of formula I as presented in Tables 1 to 9.
The pharmaceutical compositions of this invention may be administered orally, parenterally or via an implanted reservoir. Oral administration or administration by injection are preferred. The pharmaceutical compositions of this invention may contain any conventional non-toxic pharmaceutically-acceptable carriers or auxiliary agents such as adjuvants or vehicles. In some cases, the pH of the formulation may be adjusted with pharmaceutically acceptable acids, bases or buffers to enhance the stability of the formulated compound or its delivery form. The term parenteral as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, and intralesional injection or infusion techniques.
The pharmaceutical compositions may be in the form of a sterile injectable preparation, for example, as a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to techniques known in the art using suitable dispersing or wetting agents (such as, for example. Tween 80) and suspending agents.
The pharmaceutical compositions of this invention may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, and aqueous suspensions and solutions. In the case of tablets for oral use, carriers which are commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried corn starch. When aqueous suspensions are administered orally, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening and/or flavoring and/or coloring agents may be added.
Other suitable vehicles or carriers for the above noted formulations and compositions can be found in standard pharmaceutical texts, e.g. in xe2x80x9cRemington""s Pharmaceutical Sciencesxe2x80x9d, 19th ed., Mack Publishing Company, Easton, Pa., 1995. Dosage levels of between about 0.01 and about 100 mg/kg body weight per day, preferably between about 0.5 and about 75 mg/kg body weight per day of the protease inhibitor compounds described herein are useful in a monotherapy for the prevention and treatment of HCV mediated disease. Typically, the pharmaceutical compositions of this invention will be administered from about 1 to about 5 times per day or alternatively, as a continuous infusion. Such administration can be used as a chronic or acute therapy. The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. A typical preparation will contain from about 5% to about 95% active compound (w/w). Preferably, such preparations contain from about 20% to about 80% active compound.
As the skilled artisan will appreciate, lower or higher doses than those recited above may be required. Specific dosage and treatment regimens for any particular patient will depend upon a variety of factors, including the activity of the specific compound employed, the age, body weight, general health status, sex, diet, time of administration, rate of excretion, drug combination, the severity and course of the infection, the patient""s disposition to the infection and the judgment of the treating physician. Generally, treatment is initiated with small dosages substantially less than the optimum dose of the peptide. Thereafter, the dosage is increased by small increments until the optimum effect under the circumstances is reached. In general, the compound is most desirably administered at a concentration level that will generally afford antivirally effective results without causing any harmful or deleterious side effects.
When the compositions of this invention comprise a combination of a compound of formula I and one or more additional therapeutic or prophylactic agent, both the compound and the additional agent should be present at dosage levels of between about 10 to 100%, and more preferably between about 10 and 80% of the dosage normally administered in a monotherapy regimen.
When these compounds or their pharmaceutically acceptable salts are formulated together with a pharmaceutically acceptable carrier, the resulting composition may be administered in vivo to mammals, such as man, to inhibit HCV NS3 protease or to treat or prevent HCV virus infection. Such treatment may also be achieved using the compounds of this invention in combination with agents which include, but are not limited to: immunomodulatory agents, such as xcex1-, xcex2-, or xcex3-interferons; other antiviral agents such as ribavirin, amantadine; other inhibitors of HCV NS3 protease; inhibitors of other targets in the HCV life cycle such as helicase, polymerase, metalloprotease, or internal ribosome entry site (IRES); or combinations thereof. The additional agents may be combined with the compounds of this invention to create a single dosage form. Alternatively these additional agents may be separately administered to a mammal as part of a multiple dosage form.
Accordingly, another embodiment of this invention provides methods of inhibiting HVC NS3 protease activity in mammals by administering a compound of the formula I, wherein the substituents are as defined above.
In a preferred embodiment, these methods are useful in decreasing HCV NS3 protease activity in a mammal. If the pharmaceutical composition comprises only a compound of this invention as the active component, such methods may additionally comprise the step of administering to said mammal an agent selected from an immunomodulatory agent, an antiviral agent, a HCV protease inhibitor, or an inhibitor of other targets in the HCV life cycle such as helicase, polymerase, or metalloprotease. Such additional agent may be administered to the mammal prior to, concurrently with, or following the administration of the compositions of this invention.
In an alternate preferred embodiment, these methods are useful for inhibiting viral replication in a mammal. Such methods are useful in treating or preventing HCV disease. If the pharmaceutical composition comprises only a compound of this invention as the active component, such methods may additionally comprise the step of administering to said mammal an agent selected from an immunomodulatory agent, an antiviral agent, a HCV protease inhibitor, or an inhibitor of other targets in the HCV life cycle. Such additional agent may be administered to the mammal prior to, concurrently with, or following the administration of the composition according to this invention.
The compounds set forth herein may also be used as laboratory reagents. The Applicant provides for the first time compounds with a low molecular weight, that are highly active and specific against the HCV NS3 protease. Some of the present compounds may be instrumental in providing research tools for designing of viral replication assays, validation of animal assay systems and structural biology studies to further enhance knowledge of the HCV disease mechanisms.
The compounds of this invention may also be used to treat or prevent viral contamination of materials and therefore reduce the risk of viral infection of laboratory or medical personnel or patients who come in contact with such materials (e.g. blood, tissue, surgical instruments and garments, laboratory instruments and garments, and blood collection or transfusion apparatuses and materials).
Methodology
Several ways of carrying the synthesis of acyclic intermediates of compounds of formula I are disclosed in WO 00/09543 and WO 00/09558 incorporated herein by reference.
The compounds of the present invention are synthesized according to the general process illustrated in Schemes I, II and III (wherein PG is an appropriate protecting groups. [In all schemes presented below, Dxe2x80x2 has the same definition as D but is 2 to 5 atom shorter].
When the invention covers compounds of formula I wherein A is a N-substituted amide, vinyl-ACCA or homo-allyl ACCA (R1) is coupled to an appropriate amine prior to the coupling to P2. Such coupling will be readily recognized by persons skilled in the art. As will be recognized by persons skilled in the art, such amide (A) is not protected but bears any relevant substituent R5 as defined above.
The ring-closing reaction (macrocyclization) is carried out by either olefin metathesis (Scheme I) or when the linker contains a nitrogen atom, by reductive amination (Scheme II), or by peptide bond formation Scheme III.
Details of these processes are presented below:
A. Macrocyclisation via Olefin Metathesis 
Scheme I
There are several ways in which the coupling sequence can be carried out which can be easily recognized by persons skilled in the art. Starting with 4-(S)-hydroxyproline, the substituent at the 4-hydroxy can be incorporated via a Mitsunobu reaction (as described in Mitsunobu Synthesis 1981, January, 1-28; Rano et al. Tet. Lett. 1994, 36, 3779-3792; Krchnak et al. Tet. Lett. 1995, 36, 6193-6196) before or after the macrocyclization. Alternatively the assembly can be done with the required 4-(R)-hydroxy-substituted proline as disclosed in the general processes of WO 00/09543 and WO 00/09558 (see below for specific examples of these fragments).
Steps A, B, C: Briefly, the P1, P2, and P3 moieties can be linked by well known peptide coupling techniques and generally disclosed in WO 00/09543 and WO 00/09558.
Step D: The formation of the macrocycle can be carried out via an olefin metathesis using a Ru-based catalyst such as the one reported by Miller, S. J.; Blackwell, H. E.; Grubbs, R. H. J. Am. Chem. Soc. 1996, 118, 9606-9614 (a); Kingsbury, J. S.; Harrity, J. P. A.; Bonitatebus, P. J.; Hoveyda, A. H. J. Am. Chem. Soc. 1999, 121, 791-799 (b) and Huang, J.; Stevens, E. D.; Nolan, S. P.; Petersen, J. L.; J. Am. Chem. Soc. 1999, 121, 2674-2678 (c). It will also be recognized that catalysts containing other transition metals such as Mo can be used for this reaction. 
Step E: Optionally, the double bond is reduced by standard hydrogenation methods well known in the art. When Axe2x80x2 is a protected carboxylic acid, it is also deprotected appropriately.
B. Macrocyclization via Reductive Amination (for Linkers Containing N)
When the linker contains a nitrogen atom, macrocyclization is achieved by reductive amination as shown in Scheme II to obtain inhibitors of general structure II. 
Step A: Hydroboration of the double bond following Brown""s procedure (H. C. Brown and B. C. Subba Rao, J. Am. Che. Soc. 1959, 81, 6434-6437) followed by oxidation of the resulting alcohol (for example via Dess-Martin periodinate, J. Am. Chem. Soc. 1991, 113, 7277-7287) affords the corresponding aldehyde.
Step B: Hydrogenation in the presence of acid leads to the removal of the amino protecting group followed by macrocyclization via reductive amination. The P3 unit used in this synthesis is easily obtained from a variety of amino acids, such as lysine, ornithine, glutamine (after a Hofmann reaction: Ber. 1881, 14, 2725) and others; these synthetic modifications are methods well known in the art.
Step C: Optionally, the secondary amine in the linker D (formed after step D) is alkylated with alkyl halides or acetylated with alkyl or aryl acid chlorides using methodologies well known in the art to obtain inhibitors of general structure II. When Axe2x80x2 is a protected carboxylic acid, it is also deprotected appropriately.
C. Macrocyclization via Lactam Formation
Alternatively, it is understood that these macrocyclic compounds with general structure I and II can be synthesized in other ways. For example P1 and P3 can be first connected to the linker D, then coupled to P2 and the macrocyclization reaction can be a lactam formation in two possible ways as will be recognized by persons skilled in the art and as shown in Scheme III. 
Synthesis of P1
The synthesis of inhibitors with general structure I and II requires the same P1 fragments:
a) vinyl ACCA, the synthesis and resolution of which is described in WO 00/09543 and WO 00/09558 and co-pending applications Ser. No. 09/368,866 incorporated herein by reference in its entirety) or
b) homoallyl ACCA (Example 1, compound 1f).
Synthesis of P2
Some of the P2 fragments used for the synthesis of compounds of formula I are described in WO 00/09543 and WO 00/09558 and co-pending applications Ser. No. 09/368,866 incorporated herein by reference in its entirety.
Other P2 fragments are synthesized as follows:
(i) Approach from the Corresponding xe2x80x9cHetxe2x80x9d Carboxylic Acid IVb 
The synthesis is carried out according to a modified procedure in Li et al. J. Med. Chem. 1994, 34, 3400-3407. Intermediate IVa where R21=OMe (Example 7, compound 7b) is prepared as described by Brown et al. J. Med. Chem. 1989, 32, 807-826.
Step A: Intermediate IVa is coupled with heterocyclic carboxylic acids IVb under basic conditions with POCl3 to activate the carboxylate group. A variety of carboxylic acids with general structure IVb are used for the preparation of inhibitors; these are either commercially available, synthesized as shown in scheme V, VI and VII, or synthesized individually using methods described in the specific examples.
Step B: Ring-closure, followed by dehydration is achieved under basic conditions to obtain quinolines of general structure IVd.
(i.a). Synthesis of xe2x80x9cHetxe2x80x9d-carboxylic Acids of General Formula IVb
A modification of the procedure described by Berdikhina el al. Chem. Heterocycl. Compd. (Engl. Transl.) 1991, 4, 427-433 is used. 
A variety of 2-alkylaminothiazolyl-4-carboxylic acids, compounds of general structure Vc, are made using the general synthetic methodology outlined in Scheme V using thioureas (Va) with different alkyl substituents (R25=alkyl group) and 3-bromopyruvic acid. This type of condensation reaction is well known in the art. Alternatively, the P2 fragment containing the 2-amino-substituted-thiazole derivatives are synthesized from the corresponding 2-carboxyl derivative as shown in scheme VI according to the procedure of: Unangst, P. C.; Connor, D. T. J. Heterocyc. Chem. 29, 5, 1992, 1097-1100. 
Examples of this process are disclosed in WO 00/09543 and WO 00/09558.
A variety of 4-alkylthiazolyl-2-carboxylic acids, compounds of general structure VIId, is made using the general synthetic methodology outlined in Scheme VII. 
The procedure described by Janusz et al. J. Med. Chem. 1998, 41, 3515-3529 is used with modifications as described as follows: Ethyl thiooxamate (VIIa) is reacted with different xcex2-bromoketones of general structure VIIb (R24=alkyl group) to form thiazolyl carboxylic acids of general structure VIId. This type of condensation reaction is well known in the art.
A variety of alkylimidazolyl-2-carboxylic acids, compounds of general structure VIIIb, are made using the general synthetic methodology outlined in Scheme VIII. 
The procedure described by Baird et al. J. Amer. Chem. Soc. 1996, 118, 6141-6146. was used: an alkyl imidazole is deprotonated with a strong base (e.g. nBuLi) and then reacted with CO2 to form the carboxylic acid VIIIb. This type of condensation reaction is well known in the art.
4-Hydroxy-7-R21 quinolines having an imidazolyl or pyrazolyl moiety at C2 are generally prepared using the methodology outlined in Scheme IX. 
The synthesis of the key intermediate, (wherein R21=OMe) 4-benzyloxy-2-chloro-7-methoxyquinoline IXa is described in detail in Example 6 (compound 6e).
Step A: At high temperatures, a variety of imidazoles, alkyl substituted imidazoles, pyrazoles or alkyl substituted pyrazoles can be used to displace the 2-chloro moiety of compound IXa giving compounds of general structure IXb.
Step B: Upon removal of the benzyl protecting group from the 4-hydroxy moiety of the quinoline by standard hydrogenation methods, quinoline derivatives of general structure IXc are obtained.
Synthesis of P3
A variety of P3 fragments are synthesized containing the appropriate D linker extension for macrocyclization by olefin metathesis. In general P3 units containing a terminal olefin for metathesis are synthesized following the general schemes shown below (Schemes X, XI and XII).
Synthesis of Linkers in Class A
This general synthesis is used to make linkers that are all carbon based (no heteroatom) (Scheme X). 
The synthesis is performed according to the procedure of Evans et al. J. Am. Chem. Soc. 1990, 112, 4011-4030.
The starting carboxylic acids (Xa) is commercially available or is prepared by know literature procedures familiar to those skilled in the art.
Step A: The carboxylic acid Xa is activated with pivaloyl chloride and then reacted with the anion of Evans""chiral auxiliary 4(S)-4-(phenylmethyl)-2-oxazolidinone following well known chemistry (Review: D. J. Ager et al. Aldrichimica Acta 1997, 30, 3-11, and references therein) to obtain compounds of general structure Xb.
Step B: Stereoselective xcex1-azidation with trizylazide, of a chiral imide enolate such as those which would form from compounds with general structure Xb in the presence of a base like KHMDS, is also well known in the art (Review: D. J. Ager et al. Aldrichimica Acta 1997, 30, 3-11, and references therein).
Step C: Reduction of the xcex1-azide, catalyzed by SnCl2, is followed by protection of the amine formed as its t-butyl carbamate gives intermediates of general structure Xc. These reactions are also well known in the art.
Step D: Finally, the chiral auxiliary is hydrolyzed under basic conditions, such as a mixture of H2O2 with LiOH, to produce the amino acid-type linkers of general structure Xe.
Alternatively, P3 moieties having the same general structure Xe are synthesized following de procedure described by M. J. Burk et al. J. Am. Chem. Soc 1998, 120, 657-663 illustrated in Scheme XI. These compounds varied in the number of methylene units (xe2x80x94CH2xe2x80x94) along the linker (m=1 to 5) and the substitution of alkyl groups at R4, but did not contain a heteroatom. 
Step A: The monoacid compound XIb is prepared from commercially available diethyl 2-acetamidomalonate by standard ester hydrolysis under basic conditions.
Step B: Knoevenagel-type condensation between an aldehyde of general structure XIc and compound XIb in the presence of a base, such as pyridine, and acetic anhydride leads to the formation of enamide intermediate XId having the Z stereochemistry around the newly formed double bond as shown.
Step C: Regioselective and enantioselective catalytic hydrogenation of the enamide intermediate XId to the amino acid intermediate XIe is achieved using Burk""s method.
Step D: The nitrogen of the acetamido derivative XIe is then di-protected with the addition of a t-butyl carbamate substituent before the acetate group, as well as the ethyl ester, are hydrolyzed under standard basic condition to obtain P3 moieties of general structure XIf.
Synthesis of Linkers in Class B

This general synthesis is used to make linkers containing oxygen or sulfur. 
Step A: Suitably protected amino acids, such Boc-(L)-serine methyl ester, Boc-(L)-threonine methyl ester or Boc-(L)-allothreonine methyl ester, are alkylated with allyl iodide in the presence of Ag2O to give the methyl ester XIIb.
Step B: Hydrolysis of the methyl ester under standard basic conditions yields the ether-type linkers of general structure XIIc (Xxe2x95x90O).
Step C: The sulfur analog is prepared from the same starting amino acid XIIa (appropriately protected as before) and its hydroxyl group is converted to a good leaving group (such as the tosylate intermediate XId) using standard methodology well known in the art.
Step D: The tosyl moiety is subsequently displaced with the anion of thioacetate leading to the formation of the thioester intermediate XIIe by inversion of the chiral center at the xcex2-carbon.
Step E: Hydrolysis of the thioester moiety under mild basic conditions yields the free thiol XIIf.
Step F: Alkylation of the thiol moiety is easily achieved under basic conditions with allyl iodide.
Step G: Finally, the sulfide analog XIIc (X=S) are obtained after hydrolysis of the methyl ester using standard procedures.
Synthesis of R3 Fragment
Examples of synthesis of fragments wherein R3 is NHxe2x80x94R31 are disclosed in WO 00/09543.